Inter-relationship of lipids transferred by the lipid-transfer protein isolated from human lipoprotein-deficient plasma

In a previous study we demonstrated that highly purified lipid-transfer protein facilitated the transfer of triglyceride, cholesteryl ester, and phosphatidylcholine between plasma lipoproteins. It remained unclear, however, whether these lipids were transferred by independent sites on the lipid-transfer protein. To address this point, we have studied the protein-mediated transfer of triglyceride, cholesteryl ester, and phosphatidylcholine as a function of the concentration and lipid composition of donor and acceptor lipoproteins. Lipoproteins labeled in vitro, reconstituted lipoproteins of defined lipid composition, and phosphatidylcholine liposomes with or without triglyceride and/or cholesteryl ester have been used to investigate the inter-relationships of lipids transferred by the lipid-transfer protein. In studies of initial (less than or equal to 10-13%) transfer, we found that, although absolute transfer rates were affected, the ratio of cholesteryl ester to triglyceride transferred was independent of donor and acceptor lipoprotein concentrations and acceptor lipoprotein lipid composition. With reconstituted lipoproteins as donor, we demonstrated that this ratio was linearly related to the ratio of cholesteryl ester to triglyceride in the donor particle; the sum of triglyceride and cholesteryl ester transferred remained constant and independent of the lipid composition of the donor. Experiments with intact lipoproteins labeled in vitro and with small unilamellar vesicles in the presence and absence of p-chloromercuriphenylsulfonate, confirmed the interdependence of triglyceride and cholesteryl ester transfer. In contrast, under all assay conditions, no correlation was found between the amount of phosphatidylcholine transferred and the transfer of triglyceride and/or cholesteryl ester. We conclude that triglyceride and cholesteryl ester compete for transfer and that the extent of transfer for each lipid is determined by its relative concentration in the donor particle, whereas phosphatidylcholine transfer is independent of triglyceride and cholesteryl ester transfer. The data also strongly support the conclusion that lipid transfer protein promotes both the exchange and net transfer of triglyceride and cholesteryl ester and that the net transfer process proceeds by a reciprocal exchange of triglyceride and cholesteryl ester without net transfer of core lipid between lipoproteins.     

The effect of oral l-carnitine on lipoprotein composition in the Watanabe Heritable Hyperlipidemic Rabbit (Oryctolagus cuniculus)

1. We have recently reported the ability of orally administered l-carnitine to lower plasma triglyceride in the Watanabe Heritable Hyperlipidemic Rabbit (WHHL), an animal model of familial hyperlipoproteinemia. 2. In the present studies we examined the effect of l-carnitine administration upon individual lipoprotein subfractions in this animal model. 3. Carnitine feeding resulted in a reduction in very low density lipoproteins (VLDL) and high density lipoprotein (HDL). 4. Compositional analysis revealed a reduction in core triglyceride content with a concomitant increase in protein and phospholipid in VLDL and low density lipoproteins (LDL). 5. Conversely, electrophoretic mobility and apolipoprotein composition were unchanged with l-carnitine. 6. These results further demonstrate the ability of l-carnitine to modulate lipoprotein lipid composition in this animal model of familial hyperlipoproteinemia.     

Effect of nonbilayer lipids on membrane binding and insertion of the catalytic domain of leader peptidase

Biological membranes contain a substantial amount of "nonbilayer lipids", which have a tendency to form nonlamellar phases. In this study the hypothesis was tested that the presence of nonbilayer lipids in a membrane, due to their overall small headgroup, results in a lower packing density in the headgroup region, which might facilitate the interfacial insertion of proteins. Using the catalytic domain of leader peptidase (delta2-75) from Escherichia coli as a model protein, we studied the lipid class dependence of its insertion and binding. In both lipid monolayers and vesicles, the membrane binding of (catalytically active) delta2-75 was much higher for the nonbilayer lipid DOPE compared to the bilayer lipid DOPC. For the nonbilayer lipids DOG and MGDG a similar effect was observed as for DOPE, strongly suggesting that no specific interactions are involved but that the small headgroups create hydrophobic interfacial insertion sites. On the basis of the results of the monolayer experiments, calculations were performed to estimate the space between the lipid headgroups accessible to the protein. We estimate a maximal size of the insertion sites of 15 +/- 7 A2/lipid molecule for DOPE, relative to DOPC. The size of the insertion sites decreases with an increase in headgroup size. These results show that nonbilayer lipids stimulate the membrane insertion of delta2-75 and support the idea that such lipids create insertion sites by reducing the packing density at the membrane-water interface. It is suggested that PE in the bacterial membrane facilitates membrane insertion of the catalytic domain of leader peptidase, allowing the protein to reach the cleavage site in preproteins.     

Interaction of bovine serum high density lipoprotein with mixed vesicles of phosphatidylcholine and cholesterol.

The interaction of sonicated, small vesicles of egg phosphatidylcholine and cholesterol (2:1, mol/mol) with bovine high density serum lipoproteins was examined in terms of lipid transfer between both types of particles and the resulting changes in lipoprotein structure. Saturation of high density lipoprotein preparations with vesicle lipids gave final lipoprotein particles with essentially unchanged protein content and composition, unchanged cholesterylester and nonpolar lipid content, but with markedly increased phospholipid content (59% increas by weight) and moderately increased cholesterol content (20% increase by weight). The lipoproteins enriched in lipid were relatively uniform, spherical particles, 110 +/- 3.6 A in diameter (6 A larger than the original lipoproteins); they had a markedly decreased intrinsic protein fluorescence, a red-shifted fluorescence wavelength maximum, and more fluid lipid domains. These results indicate that the direct addition of excess lipids from membranes or other lipoproteins is a possible mechanism for lipid transfer to high density lipoproteins. Also they suggest a structural flexibility of high density lipoproteins that allows the addition of significant amounts of surface components.     

Characterization of subfractions of triglyceride-rich lipoproteins separated by gel chromatography from blood plasma of normolipemic and hyperlipemic humans

As judged from measurements of the diameters of particles fixed with osmium tetroxide and shadowed with platinum, gel chromatography on 2% agarose has been shown to be an effective quantitative method for separating triglyceride-rich lipoproteins according to particle size. Particles in the size range of chylomicrons, uncontaminated by lipoproteins smaller than about 700 A or by other serum proteins, emerged in the void volume of the column, and very low density lipoproteins with diameters between 400 and 700 A were separated into fractions with average standard deviation of 71 A from the mean. Systematic comparison of the relationship between diameter and chemical composition of fractions obtained from subjects with various hyperlipoproteinemic disorders demonstrated a precise correlation consistent with a spherical model for these lipoproteins in which phospholipids, free cholesterol, and protein occupy a surface monolayer with an invariant thickness of 21.5 A surrounding a liquid core of triglycerides and cholesteryl esters. The chemical composition of very low density lipoproteins of given particle size in most recognized types of hyperlipemia was similar to that of normolipemic subjects, but particles in the size range of chylomicrons sometimes had higher contents of cholesteryl esters and free cholesterol. Results obtained in subjects with dysbetalipoproteinemia were consistent with the presence of three populations of particles. Two of these, with mean diameters of about 850 and 350 A, had unusually high cholesteryl ester content and reduced triglyceride content and may represent "remnants" of the metabolism of structurally normal chylomicrons and very low density lipoproteins, respectively. The third, a heterogeneous group with intermediate range of particle size and pre-beta mobility, may represent a population of very low density lipoproteins with relatively normal composition.     

Conversion of human plasma high density lipoprotein-2 to high density lipoprotein-3. Roles of neutral lipid exchange and triglyceride lipases

Cholesterol esters accumulating in human plasma high density lipoproteins (HDL) are important in conversion of HDL3 to larger HDL2. We studied whether mechanisms of removal of cholesterol esters from HDL might be important in a reverse direction, i.e. conversion of HDL2 to HDL3. Native HDL2 or HDL3 is incubated with very low density lipoproteins (VLDL) and lipoprotein-poor plasma (d greater than 1.21 g/ml) at 37 degrees C. After incubation, "modified" (M) VLDL, and HDL2 or HDL3 are reisolated by ultracentrifugation. In modified M-HDL2 or M-HDL3, triglyceride becomes the major core lipid as the triglyceride/cholesterol ester weight ratio increases 8-10-fold relative to native HDL. With only small changes in protein/phospholipid ratios in M-HDLs, the large decrease in cholesterol ester/protein ratios suggest net cholesterol ester loss from HDL. Quantitative recovery analyses prove that the cholesterol esters lost from HDL are transferred to M-VLDL, which is now richer in cholesterol ester and poorer in triglyceride. These substantial exchanges of HDL lipids are not associated by significant transfer of HDL apoproteins but are dependent on neutral lipid transfer factors present in human lipoprotein-poor plasma (d greater than 1.21 g/ml). Similar results are obtained when purified core lipid transfer protein replaces d greater than 1.21 g/ml plasma in these incubations. After depletion of cholesterol ester from HDL, most but not all, exchanged triglyceride can be removed by lipolysis with either hepatic or lipoprotein lipase, resulting in a post-lipolysis HDL2 with an increased triglyceride content relative to normal HDL. With successive incubations with VLDL, and core lipid transfer factors, HDL2 loses more than two-thirds of its cholesterol esters. After lipolysis of acquired triglyceride, HDL2 is remodeled, in both composition and flotation parameters, toward HDL3.     

NMR studies of pig low- and high-density serum lipoproteins. Molecular motions and morphology.

1. NMR spectra of porcine high- and low density lipoproteins (d 1.120--1.210 and 1.019--1.070, respectively) and their extracted lipids were obtained as functions of temperature, frequency and solution viscosity, and from solutions to which paramagnetic species had been added. 2. About one-third of the N(CH3)3 groups in low-density lipoproteins are so immobile that they do not give a sharp resonance at any temperature up to 65 degrees C, unless the particles are disrupted with sodium dodecylsulphate. Most of the protein residues also undergo little segmental motion. 3. A marked restriction of motion of acyl chain terminal CH3 groups suggests that chain interdigitation occurs in low-density lipoprotein. Apart from this, there is a general ordering of the lipids without a decrease in the rate of rotation about bonds, suggesting that the protein organizes the lipids by controlling the molecular packing rather than by direct strong interactions. The lipids are more ordered in low-density than in high-density lipoprotein. 4. All phospholipids with mobile N(CH3)3 groups are at the particle surfaces, in patches separated by protein. In low-density lipoprotein the patches are raised proud of the protein, whereas in high-density lipoproteins the protein and lipid polar groups are coplanar. 5. The high-density lipoprotein results are consistent with literature models for the structure. The low-density lipoprotein results suggest a new model, which is basically a trilayer. The centre consists of a monolayer of phospholipid with tightly-packed polar groups in contact with a protein core. The outer monolayer of phospholipid contains the rest (most) of the protein; the central layer contains the neutral lipid (cholesterol esters and triglycerides), interdigitated into both the inner and outer monolayers. Unesterified cholesterol is distributed through all three layers.     

Enhancement of non-polar lipid transfer reaction through stabilization of substrate lipid particles with apolipoproteins

Transfer of lipids was studied between human plasma low density lipoproteins (LDL) and triolein particles coated with an egg phosphatidylcholine monolayer, with diameter of 27 +/- 4 nm. The lipid particles were unstable and seemed to aggregate to LDL when incubated with LDL either in the presence or the absence of bovine serum albumin. Human apolipoproteins A-I, A-II, C-II, C-III, and E stabilized the lipid particles and completely prevented this process. Cholesterol rapidly appeared in the lipid particles to reach homogeneous distribution among the phospholipid surfaces of LDL and the lipid particles regardless of whether apolipoproteins were present or absent. Cholesteryl ester spontaneously appeared in the lipid particles to some extent in the absence of the apolipoproteins, and human plasma lipid transfer protein enhanced this reaction only to a very limited extend. When the lipid particles were stabilized with the apolipoproteins, spontaneous cholesteryl ester transfer was minimized and the lipid transfer protein catalyzed the transfer of cholesteryl ester markedly. There was no specific difference among the apolipoproteins in stabilizing the particles and enhancing the transfer reaction. Reciprocal decrease in volume of triglyceride was observed at the same time in the lipid particles until the relative content of cholesteryl ester in the cores of LDL was the same as in the lipid particles. The kinetics of the cholesteryl ester and triglyceride transfer was consistent with the model that the reaction is bidirectional in equilibrium and takes both non-polar lipids as substrate in a single pool.     

Interaction of plasma-derived lipid transfer protein with macrophages in culture

This study investigates the ability of human plasma-derived lipid transfer protein to facilitate lipid transfer to and from intact viable cells in culture. Mouse peritoneal macrophages or J774 macrophages were preincubated with acetylated low density lipoprotein and [3H]oleate/albumin to promote the intracellular synthesis and accumulation of cholesteryl [3H]oleate and 3H-labeled triglyceride. The addition of partially purified lipid transfer protein to cultures of lipid-loaded macrophages resulted in a time and concentration-dependent transfer of radiolabeled cholesteryl ester and triglyceride from macrophages to the medium. At 48 hr, lipid transfer protein facilitated the net transfer of 16 and 11% of cellular cholesteryl ester and triglyceride radioactivity, respectively, to the medium; transfer in the absence of the lipid transfer protein was less than 2%. The transfer of cholesteryl ester radioactivity was accompanied by a similar decrease in cellular cholesteryl ester mass indicating a net transfer event. Lipid transfer from cells was not dependent on the presence of a lipoprotein acceptor in the medium; however, low and high density lipoproteins present at 200 micrograms cholesterol/ml did significantly stimulate the transfer protein-facilitated efflux of these lipids. Lipid transfer protein did not appear capable of transferring radiolabeled lipid from low density or high density lipoprotein to macrophages. Radiolabeled cholesteryl ester and triglyceride transferred from cells to the medium by lipid transfer protein were associated with large molecular weight (greater than 2 x 10(6)) components in the medium with an average density greater than 1.21 g/ml; these lipids were not associated with lipid transfer protein itself. However, these radiolabeled lipids were readily incorporated into low or high density lipoproteins when these lipoproteins were added to the medium either during or after its incubation with cells. It is concluded that lipid transfer protein can facilitate the net efflux of cholesteryl esters from intact, living macrophages. These studies suggest a novel and potentially antiatherogenic role for lipid transfer protein.     

Identification of the lipoprotein initiating domain of apolipoprotein B

We have explored the minimum sequence requirement for the initiation of apolipoprotein B (apoB)-mediated triglyceride-rich lipoprotein assembly. A series of apoB COOH-terminal truncation mutants, spanning a range from apoB34 (amino acid residues 1-1544 of apoB100) to apoB19 (residues 1-862) were transfected into COS cells with and without coexpression of the microsomal triglyceride transfer protein (MTP). ApoB34, -25, -23, -21, -20.5, and -20.1 underwent efficient conversion to buoyant lipoproteins when coexpressed with MTP. ApoB19.5 (amino acids 1-884) also directed MTP-dependent particle assembly, although at reduced efficiency. When apoB19.5 was truncated by another 22 amino acids to form apoB19, MTP-dependent lipoprotein assembly was abolished. Analysis of the lipid stoichiometry of secreted lipoproteins revealed that all apoB truncation mutants formed spherical particles containing a hydrophobic core. Even highly truncated assembly-competent forms of apoB, such as apoB19.5 and 20.1, formed lipoproteins with surface:core lipid ratios of <1. We conclude that the translation of the first approximately 884 amino acids of apoB completes a domain capable of initiating nascent lipoprotein assembly. The composition of lipids recruited into lipoproteins by this initiating domain is consistent with formation of small emulsion particles, perhaps by simultaneous desorption of both polar and neutral lipids from a saturated bilayer.     

Difference in lipid packing sensitivity of exchangeable apolipoproteins apoA-I and apoA-II: an important determinant for their distinctive role in lipid metabolism

Exchangeable apolipoproteins A-I and A-II play distinct roles in reverse cholesterol transport. ApoA-I interacts with phospholipids and cholesterol of the cell membrane to make high density lipoprotein particles whereas apolipoprotein A-II interacts with high density lipoprotein particles to release apolipoprotein A-I. The two proteins show a high activity at the aqueous solution/lipid interface and are characterized by a high content of amphipathic α-helices built upon repetition of the same structural motif. We set out to investigate to what extent the number of α-helix repeats of this structural motif modulates the affinity of the protein for lipids and the sensitivity to lipid packing. To this aim we have compared the insertion of apolipoproteins A-I and A-II in phospholipid monolayers formed on a Langmuir trough in conditions where lipid packing, surface pressure and charge were controlled. We also used atomic force microscopy to obtain high resolution topographic images of the surface at a resolution of several nanometers and performed statistical image analysis to calculate the spatial distribution and geometrical shape of apolipoproteins A-I and A-II clusters. Our data indicate that apolipoprotein A-I is sensitive to packing of zwitterionic lipids but insensitive to the packing of negatively charged lipids. Interestingly, apolipoprotein A-II proved to be insensitive to the packing of zwitterionic lipids. The different sensitivity to lipid packing provides clues as to why apolipoprotein A-II barely forms nascent high density lipoprotein particles while apolipoprotein A-I promotes their formation. We conclude that the different interfacial behaviors of apolipoprotein A-I and apolipoprotein A-II in lipidic monolayers are important determinants of their distinctive roles in lipid metabolism.     

Role of lipid transfers in the formation of a subpopulation of small high density lipoproteins

The effect of lipid transfers on the structure and composition of high density lipoproteins (HDL) has been studied in vitro in incubations that contained the lipoprotein-free fraction of human plasma as a source of lipid transfer protein. These incubations did not contain lecithin:cholesterol acyltransferase activity and were not supplemented with lipoprotein lipase. Incubations were performed at 37 degrees C for 6 hr in both the presence and absence of either added very low density lipoproteins (VLDL) or the artificial triglyceride emulsion, Intralipid. Incubation in the absence of added VLDL or Intralipid had little or no effect on the HDL. By contrast, incubation in the presence of either VLDL or Intralipid resulted in marked changes in the HDL. The effect of incubation with VLDL was qualitatively similar to that of Intralipid; both resulted in obvious transfers of lipid and changes in the density, particle size, and composition of HDL. Incubation of the plasma fraction of density 1.006-1.21 g/ml, total HDL, or HDL3 with either VLDL or Intralipid resulted in the following: 1) a depletion of the cholesteryl ester and free cholesterol content and an increase in the triglyceride content of both HDL2 and HDL3; 2) a decrease in density and an increase in particle size of the HDL3 to form a population of HDL2-like particles; and 3) the formation of a discrete population of very small lipoproteins with a density greater than that of the parent HDL3. The newly formed lipoproteins had a mean particle radius of 3.7-3.8 nm and consisted mainly of protein, predominantly apolipoprotein A-I and phospholipid.     

Progress towards understanding the role of microsomal triglyceride transfer protein in apolipoprotein-B lipoprotein assembly

The microsomal triglyceride transfer protein (MTP) is necessary for the proper assembly of the apolipoprotein B containing lipoproteins, very low density lipoprotein and chylomicrons. Recent research has significantly advanced our understanding of the role of MTP in these pathways at the molecular and cellular level. Biochemical studies suggest that initiation of lipidation of the nascent apolipoprotein B polypeptide may occur through a direct association with MTP. This early lipidation may be required to allow the nascent polypeptide to fold properly and therefore avoid ubiquitination and degradation. Concerning the addition of core neutral lipids in the later stages of lipoprotein assembly, cell culture studies show that MTP lipid transfer activity is not required for this to occur for apolipoprotein B-100 containing lipoproteins. Likewise, MTP does not appear to directly mediate addition of core neutral lipid to nascent apoB-48 particles. However, new data indicate that MTP is required to produce triglyceride rich droplets in the smooth endoplasmic reticulum which may supply the core lipids for conversion of nascent, dense apoB-48 particles to mature VLDL. In addition, assembly of dense apolipoprotein B-48 containing lipoproteins has been observed in mouse liver in the absence of MTP. As a result of these new data, an updated model for the role of MTP in lipoprotein assembly is proposed.     

Effect of saturated and unsaturated lipid on the composition of mesenteric triglyceride-rich lipoproteins in the rat

The effect of saturated and unsaturated lipids on the composition of mesenteric lymph triglyceride-rich lipoproteins was studied in rats. A short-term steady-state infusion model was developed in mesenteric lymph fistula rats. Micellar solutions of linoleate, oleate, or palmitate were infused intraduodenally. Steady-state conditions of lymph flow, triglyceride, and apoA-I and apoB secretion rates were achieved in hours 3-5 after the start of the infusion. During this steady-state period, triglyceride-rich lipoproteins were prepared and characterized. With lipid infusion there were the expected increases in secretion rates of triglyceride, apoB, and apoA-I both in whole lymph and in the d less than 1.006 g/ml lipoproteins. Compositional analysis of d less than 1.006 g/ml lipoproteins revealed no difference in the ratios of phospholipid or apoA-I (surface) to triglyceride (core) constituents between saturated and unsaturated lipids, suggesting a similar particle size. This was directly verified by agarose gel filtration and electron microscopy carried out at 27 degrees C, which showed no difference in particle size between linoleate and palmitate chylomicrons. When these lipoproteins were prepared at 4 degrees C, palmitate lipoproteins exhibited dramatically changed gel filtration elution profiles, suggesting a shift to smaller or at least distorted particles and questioning earlier results suggesting a smaller size for saturated fat d less than 1.006 g/ml lipoproteins. Despite the similarity of size between saturated and unsaturated chylomicrons, the apoB content of unsaturated linoleate chylomicrons was significantly lower than that of palmitate chylomicrons. This difference was present whether chylomicrons were prepared by centrifugation or by gel filtration. The clearance of palmitate chylomicrons from the circulation of recipient rats was slightly more rapid than that of linoleate chylomicrons. The mechanism for this apparently selective increase in the apoB content of saturated fat chylomicrons is unknown but the present studies suggest that these changes may be of physiologic significance, perhaps relating to the potential atherogenicity of saturated lipids.     

The conformation of apolipoprotein A-I in high-density lipoproteins is influenced by core lipid composition and particle size: a surface plasmon resonance study

Plasma high-density lipoproteins (HDL) are a heterogeneous group of particles that vary in size as well as lipid and apoprotein composition. The effect of HDL core lipid composition and particle size on apolipoprotein (apo) A-I structure was studied using surface plasmon resonance (SPR) analysis of the binding of epitope-defined monoclonal antibodies. The association and dissociation rate constants of 12 unique apo A-I-specific monoclonal antibodies for isolated plasma HDL were calculated. In addition, the association rate constants of the antibodies were determined for homogeneous preparations of spherical reconstituted HDL (rHDL) that contained apo A-I as the sole apolipoprotein and differed either in their size or in their core lipid composition. This analysis showed that lipoprotein size affected the conformation of domains dispersed throughout the apo A-I molecule, but the conformation of the central domain between residues 121 and 165 was most consistently modified. In contrast, replacement of core cholesteryl esters with triglyceride in small HDL modified almost the entire molecule, with only two key N-terminal domains of apo A-I being unaffected. This finding suggested that the central and C-terminal domains of apo A-I are in direct contact with rHDL core lipids. This immunochemical analysis has provided valuable insight into how core lipid composition and particle size affect the structure of specific domains of apo A-I on HDL.     

Plasma cholesteryl ester transfer protein has binding sites for neutral lipids and phospholipids

The plasma cholesteryl ester-transfer protein (CETP, Mr 74,000) promotes exchange of both neutral lipids and phospholipids (phosphatidylcholine, PC) between lipoproteins. To investigate the mechanism of facilitated lipid transfer, CETP was incubated with unilamellar egg PC vesicles containing small amounts of cholesteryl ester (CE) or triglyceride, and then analyzed by gel filtration chromatography. There was rapid transfer of radiolabeled CE or triglyceride and PC from vesicles to CETP. The CETP with bound lipids was isolated and incubated with low density lipoproteins (LDL), resulting in transfer of the lipids to LDL. The CETP bound up to 0.9 mol of CE or 0.2 mol of triglyceride and 11 mol of PC/mol of CETP. para-Chloromercuriphenylsulfonate, an inhibitor of CE and triglyceride transfer, was found to decrease the binding of radiolabeled CE and triglyceride by CETP. Under various conditions the CETP eluted either as an apparent monomer with bound lipid (Mr 75,000-93,000), or in complexes with vesicles. The distribution of CETP between these two states was influenced by the presence of apoA-I or albumin, incubation time, vesicle/CETP ratio, and buffer pH and ionic strength. The results indicate that the CETP has binding sites for CE, triglyceride, and PC which readily equilibrate with lipoprotein lipids and suggest that CETP can act as a carrier of lipid between lipoproteins.     

Effect of fasting on the composition of plasma lipoproteins in cholesterol-fed diabetic rabbits

Cholesterol-fat feeding is associated with unusual alterations in the composition of plasma lipoproteins in alloxan-diabetic rabbits. In the present study plasma lipoprotein lipid and apoprotein composition was studied before and after 48 hr of fasting in cholesterol-fed diabetic and control rabbits in order to further characterize these alterations. Compared with control rabbits, the diabetic rabbits had similar plasma cholesterol levels, but 100-fold higher triglyceride levels prior to fasting. These plasma lipids were distributed mainly to large, Sf greater than 400 plasma lipoproteins in the diabetic rabbits, and to beta-VLDL in control rabbits. Sf greater than 400 lipoproteins, VLDL, IDL, LDL, and HDL from diabetic rabbits had triglyceride as the predominant lipoprotein core lipid. Sf greater than 400 lipoproteins and VLDL from diabetic rabbits had lesser amount of apoprotein E, and greater amounts of apoproteins A-I, A-IV, and B-48 as percent of total apoprotein mass in comparison with control rabbits. Fasting reduced plasma triglyceride levels by 55% in diabetic rabbits. Sf greater than 400 lipoprotein and VLDL triglyceride content decreased but remained a major core lipid. Fasting eliminated apoproteins A-I and A-IV from Sf greater than 400 lipoproteins and VLDL, but had no significant effect on apoB-48 content. Insulin treatment of the diabetic rabbits reduced plasma triglyceride by approximately 90% resulting in cholesteryl ester-rich particles reassembling beta-VLDL both in the Sf greater than 400 lipoprotein and VLDL fractions. These results indicate that the alterations in plasma lipoproteins in cholesterol-fed diabetic rabbits result from the presence in the d less than 1.006 g/ml plasma lipoprotein class of partially metabolized, intestinally derived particles.     

Unravelling the influence of surface lipids on the structure,dynamics and interactome of high-density lipoproteins

Surface lipids influence the biological activities of high-density lipoproteins (HDLs) but their species-specific effects on HDL structure, dynamics, and surface interactome has remained unclear. Building upon the five-lipid species HDL models developed and characterised in previous work, representative models of the major HDL subpopulations found in human plasma containing apolipoprotein A-I (apoA-I) have been studied using molecular dynamics simulation to describe their varying degrees of surface lipidome complexity. Specifically, two additional sets of representative HDL subpopulation particles were developed, one with sphingomyelin (SM) and the other with SM, phosphatidylethanolamine, phosphatidylinositol, and ceramide in quantities reflecting average levels characterised for HDL subpopulations derived from normolipidemic patients. These lipid species were assessed in terms of HDL size, morphology, dynamics, and overall interactome. The findings reveal that the presence of a representative SM fraction marginally enhanced HDL interfacial curvature and surface monolayer rigidity, manifesting in tighter phospholipid packing and slower surface lipid dynamics relative to SM-deficient HDL models. Furthermore, the presence of SM resulted in a reduction in the solvent exposure of core lipids and cholesterol molecules, whilst also enhancing apolipoprotein conformational flexibility and its overall twisting across the HDL surface. The hydrophobicity of apoA-I-bound lipid patches and the proportion of apoA-I hydrophobic surface area is enhanced by the overall lipidation of apoA-I irrespective of lipid composition. These findings offer new insights into how the surface lipid composition of different HDL subpopulations can significantly impact the overall interactome of HDL particles, potentially influencing subpopulation-specific biological functions like lipid scavenging and receptor interactions.     

Interfacial Tension and Surface Pressure of High Density Lipoprotein,Low Density Lipoprotein,and Related Lipid Droplets

Lipid droplets play a central role in energy storage and metabolism on a cellular scale. Their core is comprised of hydrophobic lipids covered by a surface region consisting of amphiphilic lipids and proteins. For example, high and low density lipoproteins (HDL and LDL, respectively) are essentially lipid droplets surrounded by specific proteins, their main function being to transport cholesterol. Interfacial tension and surface pressure of these particles are of great interest because they are related to the shape and the stability of the droplets and to protein adsorption at the interface. Here we use coarse-grained molecular-dynamics simulations to consider a number of related issues by calculating the interfacial tension in protein-free lipid droplets, and in HDL and LDL particles mimicking physiological conditions. First, our results suggest that the curvature dependence of interfacial tension becomes significant for particles with a radius of ～5 nm, when the area per molecule in the surface region is <1.4 nm². Further, interfacial tensions in the used HDL and LDL models are essentially unaffected by single apo-proteins at the surface. Finally, interfacial tensions of lipoproteins are higher than in thermodynamically stable droplets, suggesting that HDL and LDL are kinetically trapped into a metastable state.     

Nascent Astrocyte Particles Differ from Lipoproteins in CSF

Abstract: Little is known about lipid transport and metabolism in the brain. As a further step toward understanding the origin and function of CNS lipoproteins, we have characterized by size and density fractionation lipoprotein particles from human CSF and primary cultures of rat astrocytes. The fractions were analyzed for esterified and free cholesterol, triglyceride, phospholipid, albumin, and apolipoproteins (apo) E, AI, AII, and J. As determined by lipid and apolipoprotein profiles, gel electrophoresis, and electron microscopy, nascent astrocyte particles contain little core lipid, are primarily discoidal in shape, and contain apoE and apoJ. In contrast, CSF lipoproteins are the size and density of plasma high-density lipoprotein, contain the core lipid, esterified cholesterol, and are spherical. CSF lipoproteins were heterogeneous in apolipoprotein content with apoE, the most abundant apolipoprotein, localized to the largest particles, apoAI and apoAII localized to progressively smaller particles, and apoJ distributed relatively evenly across particle size. There was substantial loss of protein from both CSF and astrocyte particles after density centrifugation compared with gel-filtration chromatography. The differences between lipoproteins secreted by astrocytes and present in CSF suggest that in addition to delivery of their constituents to cells, lipoprotein particles secreted within the brain by astrocytes may have the potential to participate in cholesterol clearance, developing a core of esterified cholesterol before reaching the CSF. Study of the functional properties of both astrocyte-secreted and CSF lipoproteins isolated by techniques that preserve native particle structure may also provide insight into the function of apoE in the pathophysiology of specific neurological diseases such as Alzheimer's disease.